Phagotosis by macrophages is an essential component of innate immunity. Although post-phagocytic delivery of microbes into macrophage lysosomes typically leads to their degradation, some pathogenic microorganisms survive phagocytosis and evade macrophage defense mechanisms. Listeria monocytogenes is an intracellular pathogen that survives by passing from phagosomes into cytoplasm. Activation of macrophages with interferon-g plus LPS or TNFa increases resistance to many pathogens, including L. monocytogenes. The long-term objective of these studies is to identify those features of macrophage endocytic compartments that counteract intracellular pathogens. The hypothesis to be investigated in this proposal is that increased resistance to pathogens in activated macrophages results from altered phagosome progression to lysosomes, plus localized delivery of toxic compounds into acidic, late endosome-like phagosomes. To test this, quantitative fluorometric methods will be used to measure endocytic compartment dynamics and physiology in activated and non-activated macrphages. The first specific aim is to measure rates of phagosome maturation, fusion and lysosomes, and fluid-phase solute recycling. In the second aim, biochemical and fluorescence microscopic methods will be used to measure intravacuolar pH and intracellular nitric oxide levels. Sites of peroxynitrite generation will be localized in the cytopolasm and in individual phagosomes. The third specific aim is to identify host and bacterial factors that influence escape of L. monocytogenes from phagosomes. L. monocytogenes secretes a hemolytic protein, listeriolysin O ( LLO ), which mediates bacterial passage from phagosomes to cytoplasm. Fluorescence microscopy will be used to identify the compartment permeabilized by L. monocytogenes , and to determine how this compartment is altered in activated macrophages. Features of LLO that mediate escape from phagosomes will be identified using bacteria expressing mutant and wild-type LLO. For each mutant, the compartment of escape, the efficiency of perforation and escape, and the pH of perforation will be determined. Both listericidal and nonlistericidal macrophages will be compared, as well as macrophages from mice with induced deletions for components of the nitric oxide or superoxide biosynthetic pathways. Because these studies will provide direct measurements of conditions inside the vacuolar compartments of activated macrophages, the results should improve understanding of host defense mechanism related to infecton by L. monocytogenes as well as other intracellular pathogens.